The present invention relates to a method for enhancing the specific immune response against an immunogenic compound, which comprises administering the immunogenic compound together with a poxvirus, recombinant or not.
Smallpox, a human infectious disease due to a vaccinia virus, was declared eradicated from the globe in 1980. This unique success was made possible by the availability of an effective virus-attenuated vaccine. Concurrent with the smallpox eradication and the cessation of vaccination, a new use for the vaccinia virus was proposed (Panicali and Paoletti, PNAS (1982) 79:4927). Utilizing molecular cloning techniques, it became possible to express genes from foreign pathogens in vaccinia virus providing new approaches to vaccination.
Since then, the original technology has been applied to the whole poxvirus family, including not only the vaccinia virus but also avipoxviruses such as fowlpox and canarypox. In order to address the issue of safety, a strategy was developed to genetically engineer a highly attenuated vaccinia virus such as the Copenhagen strain that would still retain the ability to induce vigorous immunological response against extrinsic antigens. A number of poxvirus constructions have been tested in clinical trials. As a matter of example, they include recombinant vaccinia and canarypoxviruses expressing Human Immunodeficiency Virus (HIV) or Plasmodium falciparum antigens. Further, it has already been proposed to combine, in an immunization protocol, a prime-administration using a recombinant poxvirus vector and booster-administrations of the purified polypeptide as encoded by the recombinant vector (See e.g., Excler and Plotkin, AIDS (1997) 11 (suppl. A): S127). Such immunization protocols are commonly referred as prime-boost protocols and are very advantageous in a number of cases, in particular for AIDS treatment or prevention.
Prime-boost protocols are however unpractical both for physicians, manufacturers and sellers, in that they require two different pharmaceutical products that have to be each identified and licensed for their specific use (priming or boost).
It has now been found that poxvirus particles may be useful as enhancer of specific immunity. Indeed, it has been observed that the immune response against a vaccinal antigen, such as an HIV or an influenza virus protein, is enhanced, when it is mixed with a poxvirus, recombinant or not. Additionally, It has also been found that an immunization protocol exclusively using a composition comprising a polypeptide and a poxvirus encoding this polypeptide, may be just as good as a prime-boost protocol. It has also surprisingly been found that the observed immunization effect is not a mere additional effect, but results from a synergism effect between the two components.
Therefore, the invention provides for:
(i) The use of a poxvirus for the manufacture of a pharmaceutical composition comprising an immunogenic compound for inducing an immune response in a vertebrate, wherein the poxvirus is able to enhance a specific immune response to the immunogenic compound.
(ii) The use of a mixture comprising (a) an immunogenic compound which comprises at least one antigenic determinant characteristic of a pathogenic microorganism or is cross-reactive with a tumor-associated antigen (TAA) and (b) a poxvirus; in the manufacture of a medicament to be administered to a vertebrate for treating or preventing an infection induced by the pathogenic microorganism or a tumoral disorder characterized by the malignant expression of the TAA; whereby said poxvirus enhances the specific immune response of the vertebrate against said immunogenic compound.
(iii) A pharmaceutical composition comprising (a) an immunogenic compound and (b) a poxvirus encoding an heterologous polypeptide which is selected from the group consisting of adhesion molecules, co-immunostimulatory molecules, apoptotic factors, cytokines, chemokines and growth hormones.
(iv) A pharmaceutical composition comprising (a) an immunogenic compound which is a first polypeptide and (b) a poxvirus encoding an heterologous polypeptide,which has an amino acid sequence identical to the amino acid sequence of the first polypeptide.
(v) A pharmaceutical composition comprising (a) an immunogenic compound which is a DNA plasmid encoding a first polypeptide and (b) a poxvirus encoding a second heterologous polypeptide, which has an amino acid sequence identical to the amino acid sequence of the first polypeptide.
(vi) A method for enhancing the specific immune response of a vertebrate to an immunogenic compound, which comprises administering to the vertebrate the immunogenic compound together with a poxvirus, whereby the poxvirus enhances the specific immune response to the immunogenic compound.
(vii) A method for treating or preventing in a vertebrate, a disorder either induced by a pathogenic microorganism or characterized by the malignant expression of a T.A.A, which comprises administering to the vertebrate, (a) an immunogenic compound which comprises at least one antigen determinant characteristic of the pathogenic microorganism or a tumor-associated antigen together with (b) a poxvirus; whereby a specific immune response to the immunogenic compound is induced in the vertebrate and whereby the poxvirus enhances the specific immune response.
(viii) A method for enhancing the specific xe2x80x9cin vitroxe2x80x9d immunostimulation of cells from an immune system against a specific immunogenic compound, which comprises (a) recovering cells from a vertebrate, (b) xe2x80x9cin vitroxe2x80x9d incubating the cells with the immunogenic compound together with a poxvirus, whereby the cells are immunostimulated against the immunogenic compound and whereby the poxvirus enhances the immunostimulation and (c) administering the immunostimulated cells obtained from step (b) to a vertebrate.
In a general manner, there exist two types of immunity: the innate immunity and the acquired immunity. The former which is phylogenetically older brings into play soluble molecules, i.a. complement factors and cells, such as NK cells or macrophages, which are innately programmed to detect noxious substances produced by pathogenic microorganisms and to provide for rapid but often incomplete antimicrobial host defense. The innate immune system intervenes as the first line of defense when an infectious agent attacks an individual. On the other hand, the innate immune system can not be educated by the antigens expressed by the pathogenic microorganisms or tumor cells during the life of an individual and in this respect; the innate immunity is confounded with the natural immunity. By contrast, the acquired immune system brings into play antigen-specific B and T lymphocyte clones the affinity of which increases by the time consecutively to repeated contacts with the specific antigen. Moreover, some of them behave as memory lymphocytes, since they have a long lasting life and are able to proliferate and expand rapidly consecutively to a further contact with a specific antigen, so that these memory lymphocytes contribute to the long term protection of an individual to infectious microorganisms. An essential goal of vaccination is to provide for these memory lymphocytes.
Accordingly, by xe2x80x9cspecific immune responsexe2x80x9d is meant a specific humoral and/or a specific cellular immune response against the immunogenic compound of the pharmaceutical composition. In the present invention, the specific humoral immune response includes both systemic and mucosal antibody responses since, to feature the humoral response, one may refer to all types of specific antibodies, i.e. IgM, all subclasses of IgG and IgA, that may be elicited by the pharmaceutical composition. The specific lymphoproliferative response and the specific cytotoxic T lymphocyte (CTL) response preferentially are the main parameters of the specific cellular immune response.
For use in the present invention, the immunogenic compound may be a chemical or a biological material that is able to induce a humoral or cellular immune response in a vertebrate. A biological material may be e.g., an attenuated, inactivated or killed virus (to the exception of a poxvirus); a bacterial strain; a pseudovirion; a bacterial extract; a capsular polysaccharides; a peptide or a polypeptide found tumor-associated, cross-reactive with a TAA or characteristic of a pathogenic agent; or a DNA plasmid encoding a peptide or a polypeptide as described above. As an example of chemical material, a hapten coupled to a carrier protein is cited.
By xe2x80x9chaptenxe2x80x9d is meant a molecule, generally of low molecular weight, which is unable to trigger an antibody response by itself, but capable, after coupling with a carrier, to induce a specific antibody response which interacts specifically with the hapten molecule. For use in the present invention, such an hapten may be a peptide which amino acid sequence is at least 5 to 6 amino acid long (minimal size of an epitope) but of low molecular weight, a chemical molecule (such as dinitrophenol), or a drug. In a particular embodiment of the present invention, a mixture according to the invention may be intended to treat drug addiction and to this end, may comprise a poxvirus, mixed with a drug, such as cocaine, coupled to a carrier molecule to induce an antibody response against the drug, in order to hamper both its fixation on the target cells, tissues or organs and the triggering of its narcotic effects. Methods of coupling a hapten to a carrier molecule are of common use for a man skilled in the art.
By xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cproteinxe2x80x9d is meant any chain of amino acids, regardless of the length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.
Advantageously, immunogenic polypeptides may be polypeptides characteristic of a pathogenic microorganism i.e. a virus, bacteria or an eucaryotic parasite, or tumor-associated antigens (that are mammalian or avian antigens which are not normally expressed; their malignant expression is characteristic of a tumoral disorder) such as tyrosinase, the MAGE protein family, the CEA, the ras protein, mutated or not, the p53 protein, mutated or not, Muc1, CEA and pSA.
For use in the present invention, immunogenic polypeptides may have amino acid sequences corresponding to the complete or partial sequence of naturally occurring polypeptides. They may also have a sequence derived by amino acid deletion, addition or substitution from the naturally occurring sequences as far as they behave as immunologic equivalents i.e., they are able to induce an immune response against the pathogenic microorganisms from which they derive or against the tumor. In other terms, an immunogenic polypeptide is also meant to include any polypeptide that is immunologically cross-reactive with a naturally occurring polypeptide found in a pathogenic agent or tumor-associated.
By xe2x80x9cimmunologically cross-reactive polypeptidesxe2x80x9d is meant polypeptides that can be recognized by antibodies, e.g. polyclonal antibodies, raised against each of the polypeptides used separately, and advantageously in a substantially purified form.
As a matter of example, the polypeptide may be an HIV antigen such as the env, gag, pol or nef protein. An HIV antigen is also meant to include any polypeptide that is immunologically cross-reactive with a naturally occurring HIV protein. For example, an HIV env protein may be the gp160 env precursor, or the gp120 or gp41 sub-unit. The gp160 precursor may be a soluble, non-cleavable precursor obtained by mutation of the cleavage site and deletion of the transmembrane region as described in U.S. Pat. No. 5,672,689. The precursor may also be truncated so that the C-terminal part of the gp41 region is removed (intracytoplasmic domain). The precursor may also be a hybrid precursor, combining in a single molecule, env sequences from various HIV strains. An HIV gag antigen may be the complete p55 precursor, the p13, p18 or p25 that naturally derive from p55, or any immunogenic gag protein fragment. In fact, a large variety of polypeptides may be substituted for the naturally occurring HIV env, gag, pol or nef proteins, yet retaining their immunogenic properties.
As an additional example the polypeptide may be an influenza peptide or polypeptide which comprises the virus envelope components such as the haemagglutinin and the neuraminidase and the virus internal components such as the protein M, the non-structural proteins and the nucleoprotein. An influenza peptide or polypeptide is also meant to include any precursor form of the mature envelope or internal proteins that are immunologically cross reactive with them. Likewise, the polypeptide or peptide may be any kind of haemagglutin or neuraminidase of the influenza virus since there are numerous antigenic variants of these two proteins.
For use in the present invention, the polypeptide characteristic of a pathogenic agent that is physically present in the composition may be purified from the pathogenic agent itself or recombinantly produced. Advantageously tumor-associated antigens (TAAs) as well will be produced by recombinant means. Standard expression vectors, promoters, terminators, etc and recombinant methods are now of common use for a man skilled in the art and recombinant expression can be readily achieved once an appropriate DNA sequence corresponding to the polypeptide is available. In a particular embodiment, polypeptides may be recombinantly produced as fusion polypeptides (i.e., a polypeptide fused through its N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail), using appropriate expression vectors, such as the pMal-c2 or pMal-p2 systems of New England Biolabs in which the peptide tail is a maltose binding protein, or the His-Tag system available from Novagen.
An immunogenic compound, e.g., a polypeptide physically present in a composition of the invention is advantageously present in a substantially purified form, i.e., it is separated from the environment in which it naturally occurs and/or is free of the majority of the polypeptides that are present in the environment in which it was synthesized.
As mentioned above, the immunogenic compound may also be a DNA plasmid unable to replicate in eucaryotic cells, comprising a DNA sequence encoding a peptide or a polypeptide, this latter being defined as herein above, under the control of an appropriate promoter which allows the peptide or polypeptide to be expressed in eucaryotic cells after transfection by the recombinant plasmid. As a matter of example, the CMV (Cytomegalovirus) early promoter is broadly used for the expression of a heterologous peptide or polypeptide in human cells transfected with DNA plasmid encoding peptide or polypeptide.
In a particular embodiment of the present invention, a DNA plasmid advantageously encodes a peptide comprising one or several epitopes characteristic of a viral, bacterial, parasitic, or tumor-associated polypeptide. As a matter of example, it is well known that tumor-associated antigens, such as Her-2 neu, are often poor immunogens, because they are essentially xe2x80x9cselfxe2x80x9d antigens. To overcome the lack of immunogenicity, it is commonly proposed to use as an immunogenic compound, instead of DNA encoding the whole polypeptide, a DNA encoding xe2x80x9csubdominantxe2x80x9d epitopes selected from the polypeptide. This strategy is also applicable to infectious microorganisms, such as HIV, Mycobacterium tuberculosis or Plasmodium falciparum for which the protective antigens are not yet defined. In a particular embodiment of the invention, aimed at the induction or the enhancement of a specific CTL response in a variety of Major Histocompatibility Complex (MHC) contexts, a pharmaceutical composition comprising a poxvirus mixed together with a DNA plasmid encoding customized peptides, may be useful. A customized peptide comprises or mimics an epitope selected throughout the whole amino acid sequence of an antigen of a pathogenic micro-organism or a tumor, as containing putative anchor motifs needed for binding to various MHC class I molecules (such as in humans, HLA-A1, HLA-A2, HLA-B7, . . . ). The customized peptides encoded by the plasmid may all together preferably trigger a specific CTL response in the main MHC contexts, of a given vertebrate.
For use in the present invention, the poxvirus may be any virus belonging to the poxviridae family. Accordingly, useful poxviruses include, capripoxvirus, suipoxvirus, molluscipoxvirus, yatapoxvirus, entomopoxvirus, orthopoxvirus and avipoxvirus; these two latter being preferred. A typical orthopoxvirus is a vaccinia virus. A suitable vaccinia virus may be e.g., the highly attenuated Copenhagen strain or the NYVAC vector that is derived from the Copenhagen strain by precise deletion of 18 open reading frames (ORFs) from the viral genome as described in Tartaglia et al, Virology (1992) 188:217. A typical avipoxvirus is a canarypoxvirus or a fowl poxvirus. A suitable canarypoxvirus may be e.g., the ALVAC vector obtained as described in Tartaglia et al (supra). A suitable fowlpox vector may be e.g., the TROVAC vector which is a plaque-cloned isolate derived from the FP-1 vaccine strain licensed for vaccination of 1 day old chicken (sold by Merial, Lyon, France) and described in Taylor et al, Vaccine (1988) 6: 497.
A poxvirus for use in the present invention may be a live, attenuated or inactivated virus. By xe2x80x9clive virusxe2x80x9d is meant a virus that is fully capable to reproduce its natural infectious cycle into sensitive cells, comprising virus entry, uncoating, gene expression, DNA replication, virus assembly, maturation and release. In a particular embodiment, a live virus may be attenuated. Attenuated virus may be obtained, e.g., by selection-of spontaneous mutants after repeated infectious cycles into sensitive cells, by selective pressure or deletion of non-essential genes using molecular biology tools. Nevertheless, whatever the process of attenuation, the viruses that are issued remain able to reproduce themselves into sensitive cells even if sometimes the spectrum of sensitive cells can decrease. As a matter of example, it may be useful to delete the vaccinia virus genome from K3L or E3L genes to render it more sensitive to the action of interferons and consequently to reduce its host restriction range (Beattie E and al., (1996) Virus Genes, 12, 89-94). As a matter of example a suitable live virus for use in humans may be a canarypoxvirus, since in human cells such a virus exhibits an abortive infectious cycle. Additionally a suitable attenuated virus for use in humans may be a NYVAC vector. By xe2x80x9cinactivated virusxe2x80x9d is meant a virus that is no more capable to reproduce its entire infectious cycle into sensitive cells as a result of either a mechanical, chemical or physical treatment. As may be easily understood, inactivation is particularly advantageous when a non-recombinant poxvirus is used.
For use in the present invention, a poxvirus may be recombinant or not. A non-recombinant poxvirus does not encode any heterologous polypeptide. On the other hand, a recombinant virus is typically a virus in the genome of which is inserted one or several foreign genes (e.g. an heterologous coding sequence located in the genome under the control of a viral promoter allowing at least a transient expression in the virus-infected cells).
A useful recombinant poxvirus encodes a heterologous peptide or polypeptide that may be of any kind. In one embodiment of the invention, the peptide or the polypeptide may be a cytokine, such as interleukin-2. (IL-2), interleukin-3 (IL-3) interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) and granulocyte macrophage-colony stimulating factor (GM CSF); a chemokine, such as RANTES (Regulated on Activation Normal T-cell Expressed and Secreted) and MCP1 (Monocyte Chemotactic protein 1); a co-immunostimulatory molecule, such as B7, CD40, CD40L and ICAMs (inter cellular adhesion molecules); an adhesion molecule; an apoptotic factor, such as p53 and TNF (tumor necrosis factor); or an hormone such as a growth hormone.
In another embodiment, the immunogenic compound for use in the present invention is a peptide or a polypeptide and the admixed poxvirus encodes a heterologous peptide or polypeptide that cross-reacts with the immunogenic compound. Accordingly, the invention also features the use of a poxvirus for the manufacture of a pharmaceutical composition comprising a first polypeptide; wherein the poxvirus encodes a second polypeptide which immunologically cross reacts with the first polypeptide. The encoded polypeptide may be the same as the one present in the composition. In other words, the encoded polypeptide has an amino acid sequence identical to that of the polypeptide present in the composition. Alternatively, the poxvirus may encode an immunogenic polypeptide that is similar to the polypeptide present in the composition, although slightly different at the amino acid sequence level. In a particular embodiment, the immunogenic polypeptide present as such in the composition originates from a particular pathogenic strain and the poxvirus vector accompanying the polypeptide encodes an allelic variant thereof i.e., the same polypeptide but from another strain. As a result, the polypeptide physically present and the encoded polypeptide may have amino acid sequences slightly different, being at least 70, 80, 90% or more identical. A composition comprising the HIV MN gp120 together with a poxvirus encoding HIV LAI gp120 is cited as a matter of example. In another embodiment, the sequences of both the polypeptide physically present and the encoded polypeptide may derive from each other by addition, deletion or substitution of one or several amino acids, provided that these polypeptides are immunologically cross-reactive. As a matter of example, it is cited a composition comprising:
(i) HIV gp160 and a poxvirus encoding HIV gp120;
(ii) HIV gp160 in a soluble and non-cleavable form and a poxvirus encoding wild-type gp160;
(iii) HIV gag p55 and a poxvirus encoding gag p18; or
(iv) HIV gp120 and a poxvirus encoding HIV gp120-p18 hybrid protein; or
(v) HIV gp120, HIV p18 and a poxvirus encoding HIV gp120-p18 hybrid protein.
As illustrated in section (v) hereinabove, a composition of the invention may comprise not only one but also two or more polypeptides present as such. The poxvirus may also encode several immunogenic polypeptides, at least one being immunologically cross-reactive with a polypeptide physically present in the composition; or the composition may contain several poxviruses. Advantageously, when several polypeptides are present as such, the compositions of the invention further contain a poxvirus that operatively encodes polypeptides, each of them being two-by-two cross-reactive with the polypeptides physically present. Alternatively, the composition may contain several poxviruses, -each of them encoding a polypeptide cross-reactive with a polypeptide physically present. As understood by a man skilled in the art, a large variety of combinations are possible.
Recombinant pox vectors may be constructed using the basic two-step technique of Piccini et al, (1987) in xe2x80x9cMeth. In Enzymologyxe2x80x9d Acad. Press, San Diego and widely used for any pox vector as described in U.S. Pat. Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587 and 5,179,993. First, the heterologous DNA sequence to be inserted into the poxvirus is placed under the control of a suitable poxvirus promoter able to direct expression of the sequence in avian or mammalian cells. The expression cassette is then introduced into an E. coli plasmid that contains a DNA region homologous to a non-essential locus of the pox vector DNA. The expression cassette is positioned so that it is flanked on both ends by poxvirus homologous DNA sequences. The resulting plasmid is then amplified by growth within E. coli and isolated. Second, the isolated plasmid containing the expression cassette to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous poxvirus DNA present on the plasmid and the viral genome gives a recombinant poxvirus modified by the presence, in a non-essential region of its genome, of the expression cassette containing the heterologous DNA sequence.
For use in the present invention, poxviruses, irrespective of whether they are recombinant or not, may be propagated on mammalian cells such as Vero cells, BHK21 cells and Chick Embryo Fibroblasts (CEF), as described in e.g., Piccini et al, and Taylor et al (supra). Once propagated, the viral particles may be merely harvested and clarified by centrifugation. They may also be purified further according to Joklick et al, Virology (1962) 18:9.
Compositions and/or methods of the invention are useful for both therapeutic and prophylactic purposes. When the immunogenic compound is characteristic of a pathogenic microorganism or a T.A.A., the specific immune response induced upon administration of the compositions or resulting from the methods of the invention, is advantageously protective against the pathogenic microorganism or the tumoral disorder. As a matter of example, there is a need to improve the current influenza vaccine which is not optimally protective in old people. Such pharmaceutical compositions or methods of the invention provide for improved protection over the flu vaccine of the prior art as exemplified in example 6.
Compositions of the invention can be manufactured in a conventional manner. In particular, the compounds can be formulated with a pharmaceutically acceptable diluent or carrier e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical diluents or carriers as well as pharmaceutical necessities for their use in pharmaceutical formulations are described in Remington""s Pharmaceutical Sciences, a standard reference text in this field.
A composition of the invention may be administered to any kind of vertebrate, i.a. to mammals or birds, in particular to humans. To this end, one can use any conventional route in use in the vaccine field e.g., via parenteral routes such as the intravenous, intradermial, intramuscular and sub-cutaneous route or mucosal routes such as nasal or oral routes. Especially, for the immunotherapy of cancer it may be useful to administer the pharmaceutical composition intratumorally or into the neighbor lymph nodes.
Compositions comprising a DNA plasmid as immunogenic compound, may advantageously be administered into the epidermis using a special device such as a gene gun or an equivalent device, or by intramuscular route. Taking into account that most of poxvirus are able to infect epidermis cells, it is worth noticing that the composition of the invention and advantageously a composition comprising a DNA plasmid mixed with a poxvirus is suitable for an intradermal or trancutaneous immunization as described by Glenn GM et al, (1998), J. Immunol. 161:3211-3214.
In a general manner, the administration can be achieved in a single dose or repeated at intervals, e.g. repeated twice or more, one or two months apart.
In compositions of the invention, the appropriate dosage of the poxvirus, and the immunogenic compound depends on various parameters understood by skilled artisans such as the vector and the immunogenic compound themselves, the route of administration, the general status of the vertebrate to be vaccinated (weight. age and the like), the type of immune response that is desired and the tumoral or infectious site. An efficient amount of the compounds is such that upon administration, an immune response against the compounds will be induced. For guidance, it is however indicated that the infectious titer (amount of virus able to infect 50% of a cell culture) per dose of the poxvirus may suitably range from 103 to 109, preferably from 105 to 108 CCID50 (Cell Culture Infectious Dose 50). The polypeptide(s) physically present in the composition may amount from 10 xcexcg to 1 mg, advantageously from 25 to 500 xcexcg, preferably from 50 to 200 xcexcg; most preferably, a single dose contains about 50-100 xcexcg of polypeptide(s). Whenever a DNA plasmid is the immunogenic compound, a convenient dose of DNA plasmid administered may amount from several ng to a few mg depending on the size of the animal giving the composition. In human beings the suitable dose of DNA plasmid per immunization may range from 20 xcexcg to 2500 xcexcg as mentioned by Wang R et al (1998), Science, 282, 476-480.
All the documents cited throughout the specification are incorporated by reference.